prickle
Planar cell polarity signaling in Drosophila requires the receptor Frizzled and the cytoplasmic proteins Dishevelled and Prickle. From initial, symmetric subcellular distributions in pupal wing cells, Frizzled and Dishevelled become highly enriched at the distal portion of the cell cortex. A Prickle-dependent intercellular feedback loop is described that generates asymmetric Frizzled and Dishevelled localization. In the absence of Prickle, Frizzled and Dishevelled remain symmetrically distributed. Prickle localizes to the proximal side of pupal wing cells and binds the Dishevelled DEP domain, inhibiting Dishevelled membrane localization and antagonizing Frizzled accumulation. This activity is linked to Frizzled activity on the adjacent cell surface. Prickle therefore functions in a feedback loop that amplifies differences between Frizzled levels on adjacent cell surfaces (Tree, 2002).
Using a glutathione-S-transferase (GST) pull-down assay, it was found that full-length, in vitro translated PkSple (one of the three alternatively spliced forms of Prickle) binds to a GST-Dsh fusion protein. To determine the domain dependency of this interaction, domains of Pk were tested for binding to individual Dsh domains. A Pk construct containing just the conserved PET and LIM domains that are common to all Pk isoforms (PkPETLIM), but not either domain individually, is sufficient for binding to full-length Dsh. Furthermore, of the three defined Dsh domains, DIX, PDZ, and DEP, it was found that PkPETLIM binds a construct containing the DEP domain but not other Dsh domains. The binding between Pk and Dsh was confirmed, and the domain specificity was verified, using a yeast two-hybrid assay. PkPETLIM fused to a DNA binding domain interacts specifically with the Dsh DEP domain but not with the other domains of Dsh. It is concluded that Pk, via its conserved PET and LIM domains, binds the Dsh DEP domain (Tree, 2002).
Since the Dsh DEP domain is required for membrane localization, it was hypothesized that Pk antagonizes Fz activity by interfering with Dsh membrane localization. To test this, it was necessary to find conditions in which Pk activity is uncoupled from Fz/Dsh activity in the adjacent cell. Fz-dependent Dsh localization was therefore reconstituted in cultured cells. In U20S cells, transfected GFP-tagged Dsh (Dsh::GFP) can be seen in punctate patches in the cytoplasm. On cotransfection with Fz, Dsh::GFP is translocated to the cell membrane, as was seen in a similar frog animal cap assay. To test whether the physical interaction between Pk and Dsh affects Dsh membrane localization, Fz, Dsh::GFP, and Pk were cotransfected. Dsh is localized in the cytoplasm in 90% of the cotransfected cells, resembling cells transfected with Dsh::GFP alone. In contrast, Pk lacking the PET domain is severely impaired in its ability to block Dsh membrane localization. Pk, therefore, interferes with Dsh membrane association in this heterologous system and by extension may interfere with Dsh membrane localization at the proximal boundary of pupal wing cells. Since Dsh is required to generate asymmetric localization of Fz, it is suggested that blocking Dsh membrane localization also inhibits the accumulation of Fz at the proximal boundary (Tree, 2002).
Fz activity on the distal side of the cell is linked to the accumulation of Pk on the proximal side of the adjacent cell and that accumulation of Pk suppresses Fz/Dsh localization at the proximal cell cortex. In this way, Fz and Pk appear to function in a feedback loop rather than a linear signaling pathway. The ordering of these members of the PCP pathway was analyzed through epistasis analysis. The loss-of-function phenotypes of pkpk-sple, dsh, and fz are very similar, suggesting that they activate the same signaling mechanism. However, these similarities make classical genetic epistasis tests difficult to interpret. In previous work, Fz overexpression phenotypes have been used to show that Fz signaling requires Dsh. These results were interpreted as implying that the PCP genes form a linear pathway with Dsh acting downstream of Fz. Consistent with this is the finding that Dsh localization requires Fz protein. In contrast, other observations do not fit this model. For example, localization of Fz requires Dsh, a presumed 'downstream' element of the pathway. Fz localization also requires the other presumed downstream elements Pk, the 7 cadherin Flamingo (Fmi), and novel TM protein Van gogh/Strabismis (Vang/Stbm) (Tree, 2002).
To clarify these results, several further epistasis tests were performed. Dsh was found to be required to generate a Fz overexpression phenotype and Fz is required to produce a Dsh overexpression phenotype. In addition, Pk is required for the Dsh and Fz overexpression phenotypes. These results argue against a simple linear pathway but are consistent with Fz, Dsh, and Pk participating in a feedback loop (Tree, 2002).
Evidence is provided that Fmi acts with Pk on the proximal side of the cell. Like Pk, overexpression of fmi using ptc-Gal4 causes hairs to point toward the midline of the wing. fmi overexpression clones show distal-domineering nonautonomy, phenocopying fz loss-of-function and pk overexpression clones. Thus, it is possible that Fmi could be acting similarly to Pk in feedback amplification of PCP signaling. Fmi is likely to be present at both sides of the cell, but it may act with Pk at the proximal side to suppress Fz signaling. However, Fmi must have an additional function since, within and surrounding fmi clones, very little Pk and Dsh localize to cell boundaries. In addition to functioning with Pk to suppress Fz/Dsh activity, it is suggested that Fmi could have a role in stabilizing the Fz complex at the membrane, without which no signaling occurs (Tree, 2002).
Whether differences in Pk activity between adjacent cells affect the localization of Dsh and Fz was examined. pk was overexpressed in the posterior of the wing (using UAS-pk, engrailed-Gal4), and Dsh and Fz localization were assessed near the edge of the pk overexpression domain. Dsh and Fz were relocalized to the antero-posterior cell boundaries, indicating that accumulation of Dsh and Fz occurs at interfaces between cells expressing high and low levels of Pk. Furthermore, within the posterior domain, Dsh and Fz are seen to accumulate to higher levels than in the anterior, consistent with a role for Pk in promoting PCP signaling. Two conclusions are drawn from these observations: (1) Dsh and Fz localization occur preferentially at boundaries between cells where one cell expresses high levels and the other expresses lower levels of Pk; (2) providing high levels of Pk amplifies Fz signaling, as measured by the increased accumulation of both Fz and Dsh at cell peripheries. In the wild-type, therefore, Pk at the proximal side of the cell drives Fz and Dsh accumulation at the distal side of the adjacent cell. Conversely, providing high levels of Fz induces higher levels of Pk accumulation (Tree, 2002).
Since Pk accumulates at boundaries between cells expressing different levels of Fz, and Dsh accumulates at boundaries between cells expressing different levels of Pk, it is suggested that Fz and Dsh on one side and Pk on the opposite side of an intercellular boundary form a self-organizing complex. Indeed, evidence is found for this in the posterior, pk-overexpressing domain of the same wings. In the posterior, Dsh and Fz accumulate in discrete patches around the cell peripheries. Simultaneous staining for Pk, Dsh, and Fmi, or Fz and Fmi, reveals that all four proteins colocalize to these patches. Thus, Pk, Dsh, Fmi, and Fz form self-organizing complexes bridging adjacent cells (Tree, 2002).
Various models have been proposed for the distribution of PCP information within Drosophila epithelia. Regardless of which model is correct, it is likely that this cue induces only a slight asymmetry within each cell. In the Drosophila wing, this initial small asymmetry may result in slightly higher levels of Fz accumulation or activation on the distal than on the proximal side of cells. However, this initial level of asymmetry is insufficient to polarize the cytoskeletal architecture of the cell and is undetectable as assessed by Dsh localization. Fz and Dsh localization subsequently become highly polarized through the action of a feedback amplification loop. Although the mechanism for such a feedback loop has not been demonstrated, it has previously been speculated that Fz activity on distal cell surfaces might promote expression of an inhibitory Wnt molecule that would suppress Fz activity on the adjacent cell surface. This study demonstrates suppression of Fz activity on adjacent cell surfaces but finds that it is mediated through asymmetric localization of Pk, which antagonizes Fz/Dsh accumulation by blocking cortical Dsh localization (Tree, 2002).
A model is proposed for a Pk-dependent feedback mechanism that functions across each P-D cell interface to amplify the difference between the initial levels of Fz/Dsh activity. Pk and Dsh are initially distributed nearly symmetrically, but in largely nonoverlapping distributions around the cells' apices. As a result of an initial, slight asymmetry in one or more components, Pk suppresses Fz accumulation on the proximal side of the cell by antagonizing Dsh cortical localization. Dsh association with the cortex is required for asymmetric Fz accumulation. Reduced proximal Fz accumulation then decreases Pk activity on the adjacent, distal side of the neighboring cell, allowing even greater Dsh cortical localization and Fz accumulation. Conversely, Pk localization is induced by Fz localization on the neighboring cell. The result is the highly asymmetric distribution of Fz, Dsh, and Pk observed at 30 hr APF. This mechanism is bidirectional, constituting a negative feedback loop that is predicted to be unstable; once any asymmetry is initiated, high levels of Fz/Dsh are promoted on one side of the boundary and low levels on the other. Fmi is proposed to play two roles. The localization of all components to the apical cell cortex depends on Fmi, which is thought to be on both sides of the boundary. Since Fmi overexpression mimics Pk overexpression, it is proposed that Fmi is activated selectively on the proximal side of the cell, where it works with Pk to suppress Fz/Dsh activity. In essence, the function of this regulatory loop depends on the balance of forces regulating Dsh membrane association on either side of the boundary. Fz recruits Dsh to the cell cortex, and Pk blocks this recruitment. Once an initial asymmetry is induced, these forces become different on either side of the boundary, and the feedback mechanism amplifies the differences (Tree, 2002).
Because fmi overexpression produces polarity phenotypes very similar to those seen with pk overexpression, it is likely that Fmi activity is also polarized across cell boundaries and participates in blocking Fz/Dsh accumulation on the distal side of the interface. However, Fmi has been proposed to exist on both sides of the boundary, and Fmi is required for the efficient localization of Fz, Dsh, and Pk to boundaries. Fmi, therefore, appears to have an additional, complex-stabilizing function. Diego (Dgo), an ankyrin repeat protein, is required for PCP signaling and localizes to P-D cell boundaries, though it has not been possible to resolve on which side of the boundary Diego is found. Dgo may function as a scaffold for assembly of PCP-signaling components (Tree, 2002).
The core PCP protein Van Gogh (Vang, also known as Strabismus [Stbm], a transmembrane protein, is likely to be involved in the feedback amplification mechanism. In a vang/stbm mutant background, Fz is localized symmetrically to the membrane in the same manner as in a pk null background, and vang/stbm has been proposed to function downstream of pk. Interestingly, in Xenopus and zebrafish, Stbm binds Dsh and seems to toggle its activity between the canonical Wnt and a PCP-like signaling pathway. Stbm antagonizes canonical Wnt signaling and activates a PCP-like pathway that regulates convergent extension and induces JNK signaling. It is proposed that both fly and vertebrate Vang/Stbm may function together with Pk, facilitating PCP signaling by antagonizing Dsh activity (Tree, 2002).
The feedback amplification mechanism described here provides a clue to understanding the long-standing problem of domineering nonautonomy. Loss-of-function clones of fz, vang/stbm, and to a lesser extent pk induce polarity phenotypes in neighboring wild-type tissue. The feedback loop model predicts that loss of Fz will disrupt the localization of these components in the neighboring distal cells, causing their polarity to reverse. Furthermore, if the immediate clone neighbors have reversed polarity, this could then cause the reversal to propagate over a distance. This phenomenon is observed near the lateral borders of a fz clone, where Pk accumulation is seen to run parallel to the clone, even at a distance from the mutant cells. Thus, the reversal in Pk localization may underlie, in part, the domineering nonauontomy observed within wild-type tissue distal to fz clones (Tree, 2002).
Once asymmetrically localized, the Fz/Dsh complex must direct reorganization of the cytoskeleton by both determining the location for prehair initiation and by limiting the number of prehairs to one. Rho and Rho-associated kinase directly regulate myosins to limit the number of prehairs. The recently described Formin homology protein, Daam1, links Dsh to Rho during vertebrate PCP-like signaling, and a Drosophila homolog may function similarly. However, further studies will be required to understand how localized Fz and Dsh orient prehair initiation (Tree, 2002).
Planar polarity decisions in the wing of Drosophila involve the assembly of asymmetric protein complexes containing the conserved receptor Frizzled. This study analyses the role of the Van Gogh/strabismus gene in the formation of these complexes and in determination of cell polarization. The Strabismus protein becomes asymmetrically localized to the proximal edge of cells. In the absence of strabismus activity, the planar polarity proteins Dishevelled and Prickle are mislocalized in the cell. Strabismus binds directly to Dishevelled and Prickle and is able to recruit them to membranes. Furthermore, the putative PDZ-binding motif at the C terminus of Strabismus is not required for its function. A two-step model is proposed for assembly of Frizzled-containing asymmetric protein complexes at cell boundaries. First, Strabismus acts together with Frizzled and the atypical cadherin Flamingo to mediate apicolateral recruitment of planar polarity proteins including Dishevelled and Prickle. In the second phase, Dishevelled and Prickle are required for these proteins to become asymmetrically distributed on the proximodistal axis (Bastock, 2003).
The subcellular localiaation of Stbm protein was investigated during wing morphogenesis using both a Stbm-YFP (Stbm yellow fluorescent protein) expressing transgene and using specific antibodies raised against Stbm. During the third instar stage, Stbm-YFP in the wing pouch localizes unevenly around apicolateral cell boundaries. Based on its molecular homology as a multi-pass transmembrane protein, it is assumed that Stbm is present in the outer cell membrane. At 18 hours of pupal life, a similar pattern is seen, Stbm-YFP still being distributed patchily in an apicolateral ring. By 24 hours, there is preferential distribution of Stbm-YFP to proximodistal cell boundaries; this distribution is clearly present at 28 hours and persists until at least 32 hours, which corresponds to the time of trichome initiation. The pattern seen with Stbm antibodies confirms that Stbm-YFP is a faithful reporter of Stbm protein distribution (Bastock, 2003).
The timecourse and distribution of Stbm broadly fits that described for other planar polarity proteins such as Fmi, Fz, Dsh and Pk-Sple. Consistent with this, good colocalization is found between Stbm-YFP and other polarity proteins. The localization of Stbm-YFP to the adherens junction zone was confirmed by costaining for Armadillo distribution. Conversely, Stbm-YFP shows no overlap with the distribution of Discs-Large, which is localized in the septate junction region. Mosaic analysis revealed that Stbm-YFP becomes preferentially distributed to the proximal edges of cells with no appreciable accumulation at distal edges (Bastock, 2003).
The three putative multipass transmembrane proteins Fmi, Fz and Stbm all play important roles in the first step of localizing planar polarity proteins to the apicolateral adherens junction zone. It is thought that Fmi acts at the top of the hierarchy in this process, since, in its absence, negligible amounts of any planar polarity proteins become apicolaterally localized. Stbm is also key, because, in its absence, both Fz and Fmi recruitment are reduced. Additionally, Stbm is also required for Dsh apicolateral recruitment and for efficient localization of Pk to membranes. Fz is not significantly required for apicolateral recruitment of Fmi, but is partly needed for apicolateral localization of Stbm and is absolutely required for apicolateral localization of Dsh. Hence, in the absence of Fmi, Fz or Stbm, one or more planar polarity proteins do not become apicolaterally localized and the process of asymmetric localization on the proximodistal axis does not occur (Bastock, 2003).
An important question is which of these factors are directly binding together, in the process of apicolateral recruitment. So far no direct protein interactions have been reported for Fmi, although it is tempting to speculate that Fmi might bind directly to Fz and Stbm in the process of apicolateral recruitment. However, Fz is able to recruit Dsh to membranes in a heterologous cell type, suggesting that these factors directly interact. In addition, vertebrate Stbm and Dsh homologs have been shown to directly interact. Direct interactions are shown between Drosophila Stbm and Dsh, and Stbm and Pk. This suggests a model in which Dsh and Pk both become apicolaterally localized as a result of direct interactions with Fz and Stbm. Notably, in the absence of Stbm, Pk accumulates in the cytoplasm, suggesting that its interaction with Stbm is important for regulating its level in the cell in addition to its subcellular localization (Bastock, 2003).
At the stage when the planar polarity proteins are apicolaterally localized, but prior to the stage when they are asymmetrically localized on the proximodistal axis of the wing, it is possible that they are present in either 'symmetric' or 'asymmetric' complexes assembled across cell-cell boundaries. If the complexes were symmetric, then Fmi, Fz, Stbm, Pk and Dsh would all be present in a complex together on the same side of the cell-cell boundary. Such symmetric complexes would then subsequently evolve into asymmetric complexes, with Fz/Dsh at distal cell edges and Stbm/Pk at proximal cell edges and Fmi on both sides. Alternatively, the initial apicolateral complexes formed could be asymmetric, with Fz/Dsh always on the opposite side of the cell-cell boundary from Stbm/Pk. These asymmetric complexes would initially be randomly oriented relative to the axes of the wing, but would gradually become aligned to the proximodistal axis. The possibility is favored that planar polarity protein complexes are initially symmetric, since Stbm directly interacts with Dsh and these molecules colocalize during earlier stages of wing development. However, it has been reported that Pk and Dsh-GFP do not precisely colocalize in early pupal wings: this observation supports the early presence of asymmetric complexes (Bastock, 2003).
After the apicolateral recruitment of planar polarity proteins, over a number of hours their localization alters such that they become asymmetrically distributed on the proximodistal axis of the wing. Although Dsh and Pk play negligible roles in the apicolateral recruitment of proteins, both are required for this subsequent proximodistal redistribution. Since overexpression of both factors leads to the accumulation of polarity proteins at apicolateral cell boundaries, it is suggested that they both function to promote the assembly and/or stabilization of protein complexes. Removal of the function of the planar polarity gene dgo also blocks asymmetric proximodistal localization but not apicolateral localization of other polarity proteins. Furthermore, overexpression of Dgo causes an accumulation of other polarity proteins at cell boundaries similar to that seen when Dsh and Pk are overexpressed. Therefore, it is proposed that Dsh and Pk act together with Dgo in the assembly of asymmetric complexes (Bastock, 2003).
Recently, it has been proposed that the function of Pk in asymmetric complex assembly is to antagonize Dsh localization to membranes. This model is mechanistically attractive, in providing an explanation for the formation of asymmetric complexes in which Dsh and Pk are found on opposite sides of cell-cell boundaries. However, it is found that in the presence of Stbm, Dsh and Pk will colocalize at the same membranes. Furthermore, it was not possible to show an effect of overexpressing Pk on the association of Fz and Dsh at membranes. In addition, high level Pk expression in vivo does not cause Dsh to lose its membrane localization but instead appears to increase levels of Dsh at the membrane. Resolution of these issues will require a more detailed understanding of the composition and properties of the protein complexes involved (Bastock, 2003).
Frizzled (Fz) signaling regulates the establishment of planar cell polarity (PCP). The PCP genes prickle and strabismus are thought to antagonize Fz signaling. They act in the same cell, R4, adjacent to that in which the Fz/PCP pathway is required in the Drosophila eye. Stbm and Pk interact physically; Stbm recruits Pk to the cell membrane. Through this interaction, Pk affects Stbm membrane localization and can cause clustering of Stbm. Pk is also known to interact with Dsh and is thought to antagonize Dsh by affecting its membrane localization. Thus the data suggest that the Stbm/Pk complex modulates Fz/Dsh activity, resulting in a symmetry-breaking step during polarity signaling (Jenny, 2003).
pk function is required in the R4 precursor, as opposed to fz PCP signaling in R3, for control of polarity establishment. Stbm, a transmembrane protein also required in R4, interacts genetically and physically with Pk. This interaction is important for the recruitment of Pk to the plasma membrane. In Xenopus animal-cap explants, Stbm and Pk relocalize each other to subdomains of the membrane. A model is proposed of how Pk/Stbm might regulate Fz/Dsh signaling activity (Jenny, 2003).
The in vitro molecular interaction between Pk and Stbm and their mutual relocalization in Xenopus animal caps suggest that they form multiprotein complexes. Several pieces of evidence indicate that the physical interaction is physiologically important: (1) correct membrane localization of Pk depends on stbm function because in stbm mutant tissue Pk staining is diffuse and absent (or strongly reduced) at the membrane; (2) Pk and Stbm interact genetically by mutually enhancing each other's GOF and LOF phenotypes in the eye; (3) pk is necessary for PCP signaling in the R4 precursor, the same cell in which stbm is required; (4) expression of the interacting domains of Pk or Stbm interferes with polarity establishment. In particular, a subfragment of 131 amino acids of the C-terminus of Pk, required for the molecular interaction, is sufficient to affect polarity (Jenny, 2003).
Both Pk and Stbm act as if they antagonize Fz signaling. (1) In zebrafish, Stbm overexpression can prevent Wnt11 from rescuing a wnt11 mutation. (2) In the Drosophila wing, overexpression of Pk leads to wing hairs pointing towards the source of the overexpressed protein, behaving like a fz LOF clone (whereas overexpression of Fz leads to hairs pointing away from the Fz source). stbm LOF clones show the opposite behavior to fz LOF clones: wing hairs point away from the mutant patch, consistent with the mutant tissue having a higher Fz-activity (Jenny, 2003).
In the Drosophila eye, evidence that pk acts antagonistically to fz comes from the fact that the Notch-signaling-responsive R4-specific reporter mdelta0.5-lacZ is expressed for a prolonged period in both R3/R4 precursors in a pksple1 mutant. This is explained if Fz activity in the R4 precursor is increased, resulting in higher levels of Dl there. This in turn leads to N activation and concomitant mdelta0.5-lacZ reporter expression in both cells of the R3/R4 pair. Conversely, in fz and dsh mutant eye discs (where Fz signaling is absent or reduced and thus Dl should not be upregulated) N-signaling activity and mdelta-lacZ expression is initially reduced in both cells. Fz activity is also antagonized by stbm in the eye. Mosaic analysis of stbm shows that it has the capability to instruct a cell to become R4 as long as the other cell of the R3/R4 pair is mutant for stbm. Therefore, in such an all-or-nothing situation, Stbm in the R3 precursor can override a positive signal of Fz, resulting in a cell fate switch to R4 fate (Jenny, 2003).
In a wild-type situation with all PCP components present in both cells, it is crucial that Stbm activity is higher in R4 than in R3 to ensure proper Fz-signaling regulation. Therefore it is an intriguing possibility that a Pk/Stbm complex in the R4 precursor ensures such higher Stbm activity, and the associated higher Fz repression there is important for a proper R3/R4 cell fate decision (Jenny, 2003).
How does the Stbm/Pk complex regulate Fz-signaling activity?
During PCP establishment in the wing, Fz, Dsh, Dgo, Fmi and Pk are initially localized uniformly around the apical circumference of wing cells. During and after PCP signaling, these proteins relocalize and become differentially enriched: Pk concentrates on the proximal side of the cell, whereas Fz and Dsh become enriched distally. Fmi becomes enriched at both sides (Jenny, 2003).
In the eye, the situation is analogous. During PCP establishment, signaling components at the R3/R4 cell border are relocalized from a uniform to a more restricted pattern. Stbm-YFP is localized on the R4 but not on the R3 side, and Fz-GFP ends up on the R3 but not the R4 side. The analogy between the R4/R3 and proximal/distal cell borders is corroborated by the genetic requirements in R3 and R4: the distally localized factors Dsh and Fz are required in R3, while proximally localized Pk is required in R4. Fmi is localized on both poles of each wing cell and also required in both cells of the R3 and R4 pair. The function of fmi has been linked to both proposed complexes, the 'Fz/Dsh side' (fmi is required for apical localization of both Fz and Dsh) and the Stbm/Pk complex. In addition to the genetic interactions between fmi and stbm or pk, a reduced membrane staining of Pk in fmi- clones in wing cells has been shown (Jenny, 2003).
How do these changes in localization occur? Localization studies in Drosophila and Xenopus suggest that Pk and Stbm influence each other's localization and form clusters in subdomains of the cell membrane. Interestingly, such Stbm/Pk complexes also affect Fz-dependent Dsh membrane localization. Thus it is an intriguing possibility that the patches observed in animal cap cells upon coinjection of Stbm with Pk represent the result of a similar, though unpolarized, symmetry-breaking step during PCP signaling (Jenny, 2003).
The PET/LIM domain of Pk can interact with the DEP-domain/C-terminus of Dsh. This interaction has been suggested to prevent Dsh membrane recruitment. Also, the C-terminus of Stbm can interact with Dsh as long as the PDZ domain is present. Since the data suggest that Pk regulates the activity and localization of Stbm, this regulation might promote or stabilize the interactions of Dsh with Stbm and/or Pk, thereby helping to pull Dsh away from a Fz-signaling complex. The Stbm/Pk complexes could then cause active release of Dsh from the membrane or target it for degradation, resulting in low levels of Dsh (and by inference Fz) at places where Pk and Stbm are enriched. Furthermore, in the R3 cell (or distally in the wing) an unknown factor might act to prevent either the formation of the Stbm/Pk complex or its effect on Dsh (Jenny, 2003).
In conclusion, Pk and Stbm form a functional complex during PCP signaling in Drosophila and during convergent extension in Xenopus. Interestingly, in zebrafish, in addition to its function in convergent extension, Stbm is also required for the caudal migration of hindbrain motor neurons. This function of Stbm is independent of Dsh and the PCP genes tested so far. It will be interesting to determine whether Stbm and Pk function together in this context as well (Jenny, 2003).
Epithelial planar cell polarity (PCP) is evident in the cellular organization of many tissues in vertebrates and invertebrates. In mammals, PCP signalling governs convergent extension during gastrulation and the organization of a wide variety of structures, including the orientation of body hair and sensory hair cells of the inner ear. In Drosophila melanogaster, PCP is manifest in adult tissues, including ommatidial arrangement in the compound eye and hair orientation in wing cells. PCP establishment requires the conserved Frizzled/Dishevelled PCP pathway. Mutations in PCP-pathway-associated genes cause aberrant orientation of body hair or inner-ear sensory cells in mice, or misorientation of ommatidia and wing hair in Drosophila. This study provides mechanistic insight into Frizzled/Dishevelled signalling regulation. The ankyrin-repeat protein Diego binds directly to Dishevelled and promotes Frizzled signalling. Dishevelled can also be bound by the Frizzled PCP antagonist Prickle. Strikingly, Diego and Prickle compete with one another for Dishevelled binding, thereby modulating Frizzled/Dishevelled activity and ensuring tight control over Frizzled PCP signalling (Jenny, 2005).
Planar cell polarity (PCP) in the Drosophila eye is established by the distinct fate specifications of photoreceptors R3 and R4, and is regulated by the Frizzled (Fz)/PCP signaling pathway. Before the PCP proteins become asymmetrically localized to opposite poles of the cell in response to Fz/PCP signaling, they are uniformly apically colocalized. Little is known about how the apical localization is maintained. Evidence is provided that the PCP protein Diego (Dgo) promotes the maintenance of apical localization of Flamingo (Fmi), an atypical Cadherin-family member, which itself is required for the apical localization of the other PCP factors. This function of Dgo is redundant with Prickle (Pk) and Strabismus (Stbm), and only appreciable in double mutant tissue. The initial membrane association of Dgo depends on Fz, and Dgo physically interacts with Stbm and Pk through its Ankyrin repeats, providing evidence for a PCP multiprotein complex. These interactions suggest a positive feedback loop initiated by Fz that results in the apical maintenance of other PCP factors through Fmi (Das, 2004).
A crucial region for PCP signaling in the eye is in rows 2-5 in the 3rd
instar larval disc behind the morphogenetic furrow (MF). Four lines of evidence support this assumption: (1) cells that take part in PCP signaling (R3/R4) are specified as photoreceptor subtypes in this region; (2) Frizzled-Notch signaling-dependent transcription in the R4 cell is initiated in this region, as detected by the mdelta0.5 reporter for the E(spl)mdelta gene; (3)
the sev-enhancer, which is active in R3/R4 cells in this region, can
drive a PCP gene in order to fully rescue the respective mutant phenotype;
and (4) in the region ahead of the MF to the first row behind it, the PCP
proteins are uniformly apically localized in all cells, before they begin at
row 2 to display the characteristic PCP protein localization pattern (Das, 2004).
Following their initial symmetric apical localization, the PCP factors
become asymmetrically enriched across the respective cell boundaries in the
proximodistal axis in the wing or the dorsoventral axis in the eye. Although
several models have been proposed as to how these complexes might be formed
and maintained, the mechanism behind the early aspect of PCP establishment
remains largely unclear. The data suggest a complex mechanism that involves
redundancy among several PCP genes (Das, 2004).
Based on the analysis of single mutant clones in the eye, only Fz and Fmi
affect PCP gene localization in a general non-redundant manner (and Stbm
affects Pk localization). The single and double mutant clone data indicate the
following (Das, 2004).
In addition to these initial requirements for apical localization and
maintenance, the subsequent asymmetric resolution of the respective PCP proteins to the R4 cell is affected and often delayed in mutant backgrounds (Das, 2004).
How is the initial apical localization of all these factors maintained? As
outlined above, none of the single mutant PCP genes, except fz and
fmi, has a significant effect on the whole complex. However, in
double mutant clones for either dgo and pk, or dgo
and stbm, localization of the PCP proteins is severely affected. Most
strikingly, the apical localization of Fmi and Fz is affected in these double
mutant combinations. In addition, the localization of Stbm and Dsh are also
affected. This could be either a direct effect of Dgo and Pk or could be mediated through their effect on Fmi [as in fmi- tissue, Stbm and Dsh
as well as Fz are reduced apically]. These data suggest that the cytoplasmic PCP proteins, which are initially recruited to the membrane by Fz (i.e. Dgo and Dsh) and
Stbm (i.e., Pk), form a protein complex that is required to maintain Fmi
apically. This interpretation is supported by the observation that Dgo physically interacts with Stbm and Pk, and thus possibly stabilizes the initial complex. Thus, these studies reveal that Dgo, Stbm and Pk are required to maintain apical Fmi localization, possibly through the physical interactions among themselves and possibly other PCP factors, during the early stages preceding PCP signaling (i.e., anterior to MF in eye). In turn, apical Fmi promotes the maintenance of an initial PCP complex at adjacent cell membranes to facilitate their signaling specific
interactions (Das, 2004).
It is possible to speculate on further implications of these data. During later stages
of PCP signaling, the localization of the PCP factors is resolved into two
types of complexes on adjacent cell membranes. The differential localization
of either Fz/Dgo or the Stbm/Pk complex in the neighboring cells (R3 versus
R4) suggests that asymmetric localization of PCP factors is maintained across
the border of the R3 and R4 cells in the eye and across proximodistal cell
borders in the wing. In the eye, the PCP proteins analyzed in this manner indeed
localize to specific sides of the R3/R4 cell border.
Similarly, proximodistal localization in the wing correlates with the
respective R3/R4-specific localization. For example, the localization of Fz
and Diego in the distal side of a wing cell correlates with the localization
on the R3 side of the R3/R4 border; conversely, Stbm localization to the
proximal side of a wing cell correlates with its localization on the R4 side
of the R3/R4 border. The localization to either the R3 or R4 side also
corresponds to the genetic requirements in either cell, as established in
mosaic analyses. Thus, since Dgo, which is initially recruited by Fz, localizes
to R3 and the pk/stbm complex localizes to R4, it is likely that at later stages during PCP signaling (posterior to MF) Fmi localization is maintained and stabilized through feedback loops on both sides of the R3/R4 boundary (Das, 2004).
A prediction from such a scenario is that Fz/Dgo are performing this
function in R3 and the Stbm/Pk complex in R4. Since
Fmi is known to function as a homophilic cell-adhesion molecule, the
removal of the feedback loop on one side could be overcome through the
homophilic recruitment of Fmi from the other side. Only when both feedback
loops are weakened on either side, can Fmi localization become affected. This is supported by the different effects of the respective double mutants posterior to the MF; those that affect both sides of the R3/R4 boundary, e.g., dgo and
stbm (R3side/R4side) or dgo and pk (R3side/R4side)
can cause Fmi delocalization, whereas double mutants affecting only one cell,
e.g., pk and stbm (both R4side), have no significant
effect (Das, 2004).
Planar cell polarity (PCP) signaling generates subcellular asymmetry along an axis orthogonal to the epithelial apical-basal axis. Through a poorly understood mechanism, cell clones that have mutations in some PCP signaling components, including some, but not all, alleles of the receptor frizzled, cause polarity disruptions of neighboring wild-type cells, a phenomenon referred to as domineering nonautonomy. A contact-dependent signaling hypothesis, derived from experimental results, is shown by reaction-diffusion, partial differential equation modeling and simulation to fully reproduce PCP phenotypes, including domineering nonautonomy, in the Drosophila wing.
This work suggests that Fz does not require a Wnt ligand in PCP signaling but that its activity is regulated by interactions between neighboring cells and differential levels of the cytoplasmic mediators Pk and Dsh. The sufficiency of this model and the experimental validation of model predictions reveal how specific protein-protein interactions produce autonomy or domineering nonautonomy (Amonlirdviman, 2005).
As the understanding of cellular regulatory networks grows, system behaviors resulting from feedback effects have proven sufficiently complex so as to preclude intuitive understanding. The challenge now is to show that enough of a network is understood to explain such behaviors. Using mathematical modeling, the sufficiency of a proposed biological model is shown and its properties studied, to demonstrate that it can explain complex PCP phenotypes and provide insight into the system dynamics that govern them (Amonlirdviman, 2005).
Many epithelia are polarized along an axis orthogonal to the apical-basal axis. On the Drosophila adult cuticle, each hexagonally packed cell elaborates an actin-rich hair that develops from the distal vertex and points distally. Genetic analyses have identified a group of PCP proteins whose activities are required to correctly polarize these arrays. The domineering nonautonomy adjacent to cell clones mutant for some, but not other, PCP genes has not yet been adequately explained. For example, in the Drosophila wing, Van Gogh/strabismus (Vang; encoding a four-pass transmembrane protein) clones disrupt polarity proximal to the mutant tissue, whereas null frizzled (fz; encoding a seven-pass transmembrane protein) alleles disrupt polarity distal to the clone. Models to explain this phenomenon have often invoked diffusible factors, referred to as factor X or Z, because they have not yet been identified. It is proposed instead that the observed behaviors of known PCP proteins are sufficient to explain domineering nonautonomy (Amonlirdviman, 2005).
Fz and other PCP signaling components accumulate selectively on the distal or proximal side of wing cells. Evidence has been provided that these proteins function in a feedback loop that amplifies an asymmetry cue, which converts uniform distributions of PCP proteins into highly polarized distributions. The proposed feedback mechanism depends on several functional relationships. Fz recruits Dishevelled (Dsh; a cytoplasmic protein) to the cell membrane. In addition, Fz promotes the recruitment of Prickle-spinylegs (Pk; a LIM domain protein) and Vang to the cell membrane of the adjacent cell. Feedback is provided by the ability of Pk (and Vang) to cell-autonomously block Fz-dependent recruitment of Dsh. This feedback loop functions strictly locally, between adjacent cells. Global directionality is imposed through the agency of the novel transmembrane protein Four-jointed and the cadherins Dachsous and Fat (Ft). Widerborst, a regulatory subunit of protein phosphatase 2A, accumulates asymmetrically within each cell and is required to bias the feedback loop. Although the mechanism by which Ft biases the direction of the feedback loop is unknown, one possibility is that Ft may direct Widerborst distribution (Amonlirdviman, 2005).
However, it is not readily apparent that this biological model does not readily explain the complex patterns observed in fields of cells containing mutant clones, and it has been argued that it cannot account for some of the observed phenotypes. Indeed, progress in understanding PCP signaling has been hampered by an inability to deduce, given a particular signaling network hypothesis, definitive links between molecular genetic interventions and tissue patterning effects. For example, although it is apparent that removing Dsh or Fz would disrupt the feedback loop, it is not obvious how the feedback loop in adjacent wild-type cells responds, such that dsh mutant clones behave autonomously, whereas for most fz alleles, mutant clones behave nonautonomously. Interestingly, though, for some fz alleles, mutant clones produce an almost cell-autonomous phenotype. As another example, Pk overexpression promotes the asymmetric accumulation of Dsh and Fz, despite the role of Pk in the feedback loop as an inhibitor of Dsh membrane recruitment (Amonlirdviman, 2005).
A mathematical model has been developed based on the described feedback loop and an initial asymmetry input representing the global directional cue. Although mathematical modeling cannot prove the correctness of the underlying biological model, the ability of the mathematical model to capture the known behaviors of the system proves the feasibility of the biological model, provides testable hypotheses, and yields insight into the factors contributing to autonomy and nonautonomy (Amonlirdviman, 2005).
The features of the biological feedback loop model have been represented as a mathematical reaction-diffusion model that describes the concentrations of Dsh, Fz, Vang, and Pk throughout a network of cells. Although the mechanisms that underlie the local feedback loop are not fully understood, the essential logic of this feedback loop is preserved by representing these interactions as binding to form protein complexes (Amonlirdviman, 2005).
Inhibition of Dsh membrane recruitment by Pk and Vang is represented in the mathematical model as an increase in the backward reaction rate of reactions in which Dsh binds Fz (or Fz complexes) by a factor dependent on the local concentration of Pk and Vang. The specific mechanism for the introduction of the directional bias into the feedback loop network is not known. Two forms of a global biasing signal were therefore implemented, and the results using either of these models were similar. The resulting mathematical model consists of a system of 10 nonlinear partial differential equations. With a given set of model parameters, an array of cells could then be simulated, and the resulting hair pattern assigned on the basis of the final distribution of Dsh (Amonlirdviman, 2005).
The model parameters, including the initial protein concentrations, reaction rates, and diffusion constants, were not known, and so these parameters were identified by constraining them to result in specific qualitative features of the hair pattern phenotypes. A sensitivity analysis showed that the model results are not highly sensitive to the precise parameter values and suggests that the conclusions regarding the feasibility of the model are valid for considerable ranges of parameters (Amonlirdviman, 2005).
In simulated wild-type cells, Dsh and Fz localize to the distal membrane, and Vang and Pk localize to the proximal membrane, as is seen in vivo. Simulated clones of cells lacking fz function disrupt polarity in wild-type cells distal to the clones, whereas simulated clones lacking Vang function disrupt polarity on the proximal side of the clones. Simulated clones lacking dsh function result in the disruption of polarity within the mutant cells, but only show a mild effect outside of the clones. The nearly, though not fully cell autonomous, phenotype is similar to that which is observed experimentally. Clones lacking all pk function show only a subtle phenotype. Examination of protein distributions shows that the results are highly concordant with published observations. Similarly, simulated overexpression clones produce results closely mimicking observed experimental results. In simulations and in wings, relatively small clones lacking a global biasing signal show no phenotype, demonstrating that not all cells need to respond to the global directional signal for the feedback loop to cooperatively align all of the cells (Amonlirdviman, 2005).
Previously, it was found that Pk overexpression in the posterior wing domain enhances the accumulation of Fz and Dsh at cell boundaries, despite the observed ability of Pk and Vang to block Dsh recruitment. Consistent with these results, Dsh and Fz are seen in a simulation of this experiment to accumulate to higher levels in the region overexpressing pk than in the wild-type region, and they accumulate perpendicular to the wildtype orientation near the anterior-posterior boundary (Amonlirdviman, 2005).
The results suggested a mechanistic explanation for the difference between autonomous and nonautonomous fz alleles. Because the nearly autonomous fz alleles (fzJ22 and fzF31) have phenotypes similar to dsh clones, it is hypothesized that these alleles may be selectively deficient in complexing with Dsh, but normal in their ability to complex with Vang. Simulations of clones in which the interaction was disrupted between Dsh and Fz by reducing the corresponding forward reaction rates produced nearly cell autonomous polarity phenotypes (Amonlirdviman, 2005).
This hypothesis makes two easily testable predictions. (1) Fz autonomous proteins should be present in the membrane and should recruit Vang to the adjacent membrane, whereas Fz nonautonomous protein should not recruit Vang. It has previously been shown that GFP-tagged FzJ22, expressed in a wild-type background, is present at the apical cell cortex, but remains symmetrically distributed, a distribution in accordance with the simulation of this condition. Examining this further, it was found that in cells adjoining clones of the autonomous fzF31 allele, Vang is recruited to the boundary between wild-type and mutant cells, whereas substantially less Vang is recruited to those boundaries in cells adjoining clones of the nonautonomous fzR52 allele. Thus, Fzautonomous proteins recruit Vang to the opposing cell surface, whereas nonautonomous alleles do not. (2) Autonomous Fz proteins should fail to recruit Dsh. Indeed, it was found that both are substantially impaired in Dsh recruitment, though somewhat less impaired than the very strong, nonautonomous fzR52 allele. Thus, strong fz alleles, many of which fail to accumulate Fz protein, display no or severely impaired interaction with Dsh and Vang, whereas autonomous alleles have impaired interaction with Dsh, but retain substantial ability to recruit Vang to the adjacent membrane. Notably, simulated overexpression of Fz with impaired Dsh interaction also produced the correct polarity disruption in cells proximal to the clones (Amonlirdviman, 2005).
The Dsh1 protein produces nearly autonomous clones, and it carries a mutation in its DEP domain, which is required for membrane localization; autonomous fz alleles bear point mutations in the first cytoplasmic loop, suggesting these mutations may affect the same interaction. A low affinity interaction between the Dsh PDZ domain and a sequence in the cytoplasmic tail of Fz has been demonstrated. These data suggest that sequences in the Dsh DEP domain, and in the Fz first intracellular loop, are also important for Dsh membrane association. Thus, a regulated, bipartite, high affinity association of Dsh with Fz may be selectively disrupted in fzautonomous alleles (Amonlirdviman, 2005).
The ability of the mathematical model to simultaneously reproduce all of the most characteristic PCP phenotypes demonstrates the feasibility of the underlying biological model as a PCP signaling mechanism. Further, the mathematical model demonstrates how the overall scheme of the model -- a local feedback loop between adjacent cells amplifying an initial asymmetry -- can explain the autonomous and nonautonomous behavior of PCP mutant clones. Alternative models invoking diffusible factors have not been supported by the identification of such factors, and the contact-dependent intercellular signaling model more readily accounts for the slight nonautonomy of dsh and fzautonomous clones than do the diffusible factor models (Amonlirdviman, 2005).
The ability of the mathematical model to make predictions and provide a detailed picture of PCP signaling is limited by the lack of complete biological understanding. Although the validity of quantitative model predictions is subject to its assumptions and the set of features used in parameter identification, the model has allowed a direct connection of a biological model to the complex behaviors it is hypothesized to explain and to explore the implications of variations in the model (Amonlirdviman, 2005).
The planar polarization of developing tissues is controlled by a conserved set of core planar polarity proteins. In the Drosophila pupal wing, these proteins adopt distinct proximal and distal localizations in apicolateral junctions that act as subcellular polarity cues to control morphological events. The core polarity protein Flamingo (Fmi) localizes to both proximal and distal cell boundaries and is known to have asymmetric activity, but the molecular basis of this asymmetric activity is unknown. This study examined the role of Fmi in controlling asymmetric localization of polarity proteins in pupal wing cells. Fmi was found to interact preferentially with distal-complex components, rather than with proximal components, and evidence is presented that there are different domain requirements for Fmi to associate with distal and proximal components. Distally and proximally localized proteins cooperate to allow stable accumulation of Fmi at apicolateral junctions, and evidence is presented that the rates of endocytic trafficking of Fmi are increased when Fmi is not in a stable asymmetric complex. Finally, evidence is provided that Fmi is trafficked from junctions via both Dishevelled-dependent and Dishevelled-independent mechanisms. A model is presented in which the primary function of Fmi is to participate in the formation of inherently stable asymmetric junctional complexes: Removal from junctions of Fmi that is not in stable complexes, combined with directional trafficking of Frizzled and Fmi to the distal cell edge, drives the establishment of cellular asymmetry (Strutt, 2009).
The differing ability of overexpressed Fmi to modulate Fz:Dsh and Stbm:Pk levels at junctions could be explained by a number of mechanisms. One likely hypothesis is that Fmi may require a cofactor for a robust interaction with Stbm, and that this cofactor is limiting when Fmi is overexpressed. Alternatively, Fmi may require posttranslational modification or a conformational change to interact with Stbm, and a factor needed for this modification is limiting. The cytoplasmic C-terminal tail of Fmi is a likely region to mediate an interaction with Fz:Dsh or Stbm:Pk; therefore, a truncated form of Fmi was constructed, in which this region is either absent or replaced with GFP (Strutt, 2009).
When overexpressed in pupal wing cells, FmideltaIntra is much more efficient at recruiting Fz and Dsh to junctions than full-length Fmi, an effect similar to that caused by removal of stbm or pk. Stbm is still reduced at junctions, although less than when full-length Fmi is overexpressed. This suggests that the C-terminal intracellular domain of Fmi is dispensible for the interaction of Fmi with Fz:Dsh and, importantly, that Fz:Dsh no longer have to compete with Stbm:Pk for access to Fmi (Strutt, 2009).
Interestingly, two isoforms of Fmi have been identified, one of which contains a PDZ binding motif (PBM) at its C terminus. It is possible that loss of the PBM alone could account for the failure of overexpressed Fmi or FmideltaIntra to associate with Stbm:Pk. However, this is unlikely, because Fmi that lacks the PBM can rescue the planar polarity phenotype of fmi mutants (Strutt, 2009).
Endogenous Fmi is thought to be localized on both proximal and distal cell boundaries. This was confirmed by expressing CFP-tagged Fmi at physiological levels in clones in pupal wings, and it was observed that levels of staining appear similar at each end of the cell, consistent with the homophilic-interaction model. Notably, expression of a GFP-tagged form of FmideltaIntra results in its preferential localization to distal cell edges, where Fz and Dsh also localize (Strutt, 2009).
Interestingly, junctional localization of FmideltaIntra-EGFP is not dependent on endogenous, full-length Fmi, suggesting that this molecule is still able to participate in homophilic interactions. Hence, the ability of FmideltaIntra-EGFP to functionally rescue the polarity phenotype of fmi null mutant clones was investigated. If FmideltaIntra-EGFP interacts preferentially with the distal Fz:Dsh complex, then Stbm recruitment to junctions inside clones would be compromised. Consequently, FmideltaIntra-EGFP:Fz complexes inside the clone would preferentially interact with Fmi:Stbm outside the clone, leading to a reversal in polarity on proximal clone edges. Importantly, this prediction is upheld, and fmi clones rescued with FmideltaIntra-EGFP exhibit weak proximal polarity inversions, such that trichomes point away from the clone, and polarity proteins are recruited to the clone boundary (Strutt, 2009).
Nevertheless, Stbm localizes asymmetrically inside the clone, although not always at the correct site, whereas in a fmi null mutant it lacks any asymmetric localization. Thus, FmideltaIntra-EGFP must retain some ability to interact with Stbm. To confirm this, the ability of full-length Fmi or FmideltaIntra-EGFP to interact with Fz and Stbm in Drosophila S2 cells was analyzed. In this assay, Fmi and FmideltaIntra-EGFP are recruited to sites of cell contact, as a result of homophilic interactions between their extracellular domains. Cotransfection of Fz or Stbm with either full-length Fmi or FmideltaIntra-EGFP in Drosophila S2 cells results in the recruitment of both to sites of cell contact (Strutt, 2009).
Interestingly, if S2 cells were transfected with either Fz or Stbm and then mixed, weak recruitment is also observed to sites of cell contact, arguing that their extracellular domains can interact independently of Fmi. Nevertheless, recruitment was weaker and less frequent than when Fmi was cotransfected, suggesting that Fmi:Fmi interactions are more important than Fz:Stbm interactions in stabilizing complexes between adjacent cells (Strutt, 2009).
The data suggest that Fz:Dsh and Stbm:Pk complexes differ in their ability to associate with Fmi. Whereas endogenous levels of Fmi result in the formation of asymmetric complexes with Fz:Dsh on one side of the boundary and Stbm:Pk on the other, overexpressing Fmi favors Fz:Dsh recruitment. Furthermore, a C-terminally deleted form of Fmi preferentially localizes distally with Fz, and overexpression of this form has an even greater preference for Fz:Dsh recruitment. Thus, the C terminus of Fmi is important in promoting the interaction with Stbm:Pk. The Fmi truncation data could be explained simply by the possibility that the C terminus of Fmi contains a direct binding site for Stbm; however, this fails to explain why overexpressed full-length Fmi prefers to recruit Fz:Dsh. It is therefore proposed that the association of Fmi with Stbm:Pk requires a limiting factor that is saturated by Fmi overexpression. The most plausible hypothesis is a requirement for a cofactor for Stbm:Pk binding, but other possibilities include saturation of the machinery for a posttranslational modification or a conformational change in Fmi (Strutt, 2009).
The data also suggest that Fmi itself needs to associate with both proximal and distal components in order to be stably localized to apicolateral junctions. Although it can form homophilic dimers between adjacent cell membranes in tissue culture, in pupal wings Fmi does not localize strongly to apical junctions and presumably fails to form stable homodimers in trans. Fz on one side of the junction and Stbm:Pk on the opposite side stabilize Fmi at junctions, most likely by promoting homophilic interactions or preventing internalization. However, Fmi appears to be capable of forming complexes with either distal or proximal components alone, but these complexes (particularly the proximal complex) are apparently less stable at junctions. Taken together with overexpression experiments, this would suggest that the most stable configuration is Fz:Fmi on one side of the boundary and Fmi*:Stbm:Pk on the other (where Fmi* denotes the modified form able to preferentially associate with Stbm:Pk) (Strutt, 2009).
In order for an asymmetric complex to be stabilized across junctions, the extracellular domains must somehow 'look' different. One possibility is that the Fz and Stbm extracellular loops interact - a view supported by S2 cell data. Alternatively, the Fmi extracellular domain, when associated with either Fz or Stbm:Pk, could undergo a conformational change that promotes homophilic Fmi interactions (Strutt, 2009).
An intriguing question is why clones of cells that overexpress Fmi behave like fz loss-of-function clones. It is suggested that within the clones, excess Fmi associates with the entire available pools of both Fz and Stbm. However, there is still a pool of uncomplexed Fmi that can associate with Fmi:Fz in adjacent wild-type cells, forming the relatively stable Fmi-Fmi:Fz configuration, thus causing polarity to be reversed on distal clone boundaries. In support of this model, an identical nonautonomous effect is seen when FmideltaIntra is overexpressed, which itself interacts only poorly with Stbm but presumably can interact with Fmi:Fz in adjacent cells outside the clone (Strutt, 2009).
Interestingly, Fmi accumulates in excess at junctions in a dsh, stbm double mutant, whereas Fz does not. Thus, although Fz acts to stabilize Fmi at junctions, Fmi does not always need to associate with Fz in a stoichiometric fashion in order to be stabilized. Perhaps as long as there is some Fz associated with Fmi, this may permit local stabilization of other Fmi molecules in cis. Alternatively, this excess accumulation of Fmi might simply represent
'unstable' Fmi homodimers that are no longer being removed from junctions by the actions of Dsh and Stbm (Strutt, 2009).
The composition of the complex with which Fmi is associated appears to be critical for determining the frequency and manner by which Fmi is turned over from the plasma membrane. Most compellingly, Fmi accumulates more strongly in an enlarged endosomal compartment in Rab7TN mutant tissue when stbm and fz are absent than when they are present. Thus, it is suggested that more Fmi is resident in the endocytic pathway when it is unable to form stable asymmetric complexes. Fmi:Fz puncta have been observed that are selectively trafficked to distal cell edges. In the current experiments, these puncta colocalize with YFP-Rab4, suggesting that Fmi and Fz are recycled back to the plasma membrane by a Rab4-dependent mechanism. Furthermore, the increased intracellular and junctional levels of Fz and Fmi in dor mutant clones suggests that in addition to being recycled to the plasma membrane, a significant fraction of internalized Fmi and Fz is also sent for degradation. It is formally possible that the intracellular accumulation of Fmi and Fz seen when lysosomal trafficking is blocked by loss of Rab7 or in dor clones is due to their being sent for degradation immediately after synthesis (e.g., if damaged or misfolded); however this is unlikely because newly synthesized Fmi-ECFP appears first at junctions before been seen in puncta (Strutt, 2009).
Stbm has not been observed in large intracellular puncta, but it seems likely that it is also internalized and recycled, possibly together with Fmi, although it must do so by alternative pathways involving smaller or more rapidly recycling particles that are not visible by confocal microscopy. Indeed, the data suggest a potential role for Dsh and Stbm in regulating junctional levels of Fmi. A stbm mutant alone results in a loss of Fmi from junctions, consistent with a need for Stbm in stabilizing Fmi in asymmetric complexes. In contrast, loss of Dsh and Stbm together increases Fmi levels at junctions, suggesting a role for Stbm in internalization. It is suggested that the outcome of any interaction of Stbm with Fmi is dependent upon whether Fmi is able to form stable homodimers with Fz on the opposite cell membrane. In a wild-type situation, one could envisage that Fmi forms stable homodimers in a Fz:Fmi-Fmi*:Stbm configuration, and that both Dsh and Stbm promote internalization of any Fmi that is not in this configuration, the majority of which is subsequently recycled back to the plasma membrane. In dsh mutants, there is reduced internalization, but the effect on Fmi levels is subtle; Fz and Stbm are still present to promote Fmi homodimer formation, and Stbm still promotes internalization of any unstable Fmi. In contrast, in stbm mutants, the number of less stable Fmi complexes (associating only with Fz) is greatly increased, favoring internalization by Dsh. Finally in dsh, stbm double mutants, Fmi is again less stable (associating only with Fz), but there is no Dsh- or Stbm-mediated internalization, leading to an overall increase of Fmi at junctions (Strutt, 2009).
How do Dsh and Stbm regulate Fmi levels at junctions? Stbm contains potential interaction motifs for the endocytic adaptor AP2, but their role has not been functionally tested. In addition, in vertebrate Wnt signaling, there is evidence that Dsh interacts with the endocytic adaptor protein β-arrestin and mu2 subunit of AP2 to mediate Wnt/Fz endocytosis and downregulation of Wnt signaling. Interestingly, in planar polarity this is no evidence that Dsh directly mediates internalization of Fz, but the data rather point to Dsh promoting Fmi internalization when it is not associated with Fz. Instead, the trafficking of Fmi together with Fz into the lysosomal pathway is Dsh independent (Strutt, 2009).
In summary, it is proposed that a number of mechanisms exist by which Fmi contributes to the generation of asymmetry at the molecular level. First, the characterization of the previously inferred asymmetry in Fmi activity indicates that Fmi normally prefers to bind to Fz and requires a limiting factor for association with Stbm:Pk. Second, Fmi stability at junctions is dependent on both Fz and Stbm:Pk, with the most stable form being Fz:Fmi bound to Fmi*:Stbm. Finally, it is proposed that entry of Fmi into the endocytic trafficking pathway is decreased if it is in a stable complex, and this is regulated either by Dsh and Stbm or independently of Dsh and Stbm, depending on whether it is associated with Fz (Strutt, 2009).
An outstanding question is how these mechanisms translate into cellular asymmetry, such that in any particular cell, heterophilic polarity complexes preferentially form with Fz:Dsh at the distal junctions, rather than having heterophilic complexes in both orientations. It is thought that the acquisition of cellular asymmetry is likely to be driven by directional trafficking of Fmi:Fz, although other models, such as a mechanism for preferential stabilization of Fmi:Fz interactions at the distal cell edge, are also possible. In addition, it seems likely that an amplification mechanism would be required, although the molecular mechanisms remain to be elucidated (Strutt, 2009).
While this manuscript was in preparation, another manuscript was published, in which Fmi was proposed to mediate an asymmetric and instructive signal between proximal and distal complexes to generate asymmetry, and thus does not act merely as a scaffold for Fz:Stbm interactions across membranes. It is argued that the current data do not provide evidence for a specific signaling function of Fmi. Instead, the hypothesis is favored that the composition of the proximal and distal complexes is distinct, and that heterophilic complexes are inherently more stable than homophilic complexes. Together, removal of unstable nonasymmetric complexes through increased endocytic turnover, in concert with directional trafficking and an unknown amplification mechanism, may be sufficient to generate asymmetry without the need to invoke a specific signaling function for any components of the complexes (Strutt, 2009).
The planar coordination of cellular polarization is an important, yet not well-understood aspect of animal development. In a screen for genes regulating planar cell polarization in Drosophila, Rab23, encoding a putative vesicular trafficking protein, was identified. Mutations in the Drosophila Rab23 ortholog result in abnormal trichome orientation and the formation of multiple hairs on the wing, leg, and abdomen. Rab23 is required for hexagonal packing of the wing cells. Rab23 is able to associate with the proximally accumulated Prickle protein, although Rab23 itself does not seem to display a polarized subcellular distribution in wing cells, and it appears to play a relatively subtle role in cortical polarization of the polarity proteins. The absence of Rab23 leads to increased actin accumulation in the subapical region of the pupal wing cells that fail to restrict prehair initiation to a single site. Rab23 acts as a dominant enhancer of the weak multiple hair phenotype exhibited by the core polarity mutations, whereas the Rab23 homozygous mutant phenotype is sensitive to the gene dose of the planar polarity effector genes. Together, these data suggest that Rab23 contributes to the mechanism that inhibits hair formation at positions outside of the distal vertex by activating the planar polarity effector system (Pataki, 2010).
The formation of properly differentiated organs often requires the planar coordination of cell polarization within tissues, a feature referred to as planar cell polarity (PCP) or tissue polarity. Although planar polarity is evident in many vertebrate tissues (such as fish scales, bird feathers, and cochlear epithelium) and it has recently been shown that PCP regulation is highly conserved throughout the animal kingdom, such polarization patterns are best studied in Drosophila melanogaster. PCP in flies is manifest in the mirror-image arrangement of ommatidia in the eye, in the adult cuticle, which is decorated with parallel arrays of hairs and sensory bristles, and in the wing, which is covered by distally pointing hairs (or trichomes). Wing hairs form during the pupal life when each cell produces a single microvillus-like prehair stiffened by actin and microtubules. In wild-type wing cells prehairs form at the distal vertex of the cells and extend distally as they grow (Pataki, 2010).
Mutations in PCP genes result in abnormal wing hair polarity patterns and wing hair number. On the basis of their cellular phenotypes (i.e., prehair initiation site and number of hairs per cell), initial studies placed PCP genes into three groups: the first group (often called the core group) includes frizzled (fz), dishevelled (dsh), starry night (stan) (also known as flamingo), Van Gogh (Vang) (also known as strabismus), prickle (pk), and diego (dgo); the second group consists of inturned (in), fuzzy (fy), and fritz (frtz) (referred to as planar polarity effectors or In group); whereas the third group includes multiple wing hairs (mwh). Double mutant analysis demonstrated that these phenotypic groups also represent epistatic groups, and it was proposed that the PCP genes may act in a regulatory hierarchy, where the core group is on the top, whereas the In group and mwh are downstream components. Subsequent work identified several other PCP genes as well. Some of these have been placed into the Fat/Dachsous group, while another group consists of cytoskeletal regulators, including Rho1 and Drok. Genetic analysis of these two groups has led to models in which the Fat/Dachsous group acts upstream of the core proteins, while Rho1 and Drok act downstream of Fz. Although the existence of a single, linear PCP regulatory pathway is debated, it is clear that in the wing, PCP genes regulate (1) the number of prehairs, (2) the place of prehair formation, and (3) wing hair orientation (Pataki, 2010).
While the molecular mechanism that restricts prehair formation to the distal vertex of the wing cells is elusive, it has been well established that the core PCP proteins adopt an asymmetrical subcellular localization when prehairs form, which appears to be critical for proper trichome placement. In addition, it has recently been found that the In group of proteins and Mwh also display an asymmetrical pattern with accumulation at the proximal zone. These studies concluded that the core PCP proteins are symmetrically distributed until 24 hr after prepupa formation (APF), when they become differentially enriched until prehair formation begins at 30-32 hr APF. This transient asymmetric localization ends by 36 hr APF. It has recently been shown that Fz and Stan containing vesicles are transported preferentially toward the distal cell cortex in the period of 24-30 hr APF, and hence, polarized vesicular trafficking might be an important determinant of PCP protein asymmetry. Other recent studies, however, challenged the view that PCP protein polarization is limited to 24-32 hr APF. Instead, it has been suggested that at least a partial proximal-distal polarization is already evident at the end of larval life and during the prepupal stages (6 hr APF). Polarity is then largely lost at the beginning of the pupal period, but becomes evident again in several hours until hair formation begins. Thus, molecular asymmetries are clearly revealed during wing hair formation, yet the molecular mechanisms that contribute to the establishment of these asymmetrical patterns are not well understood (Pataki, 2010).
In a large-scale mosaic type of mutagenesis screen, Drosophila Rab23, encoding a vesicle trafficking protein, was identified as a PCP gene involved in the regulation of trichome orientation and number in various adult cuticular structures, including the wing, abdomen, and leg. This study shows that Rab23 plays a modest role in cortical polarization of the core PCP proteins in the wing and that Rab23 associates with at least one core protein, Pk. Additionally, it was found that Rab23 contributes to the mechanism that restricts actin accumulation and thus, prehair initiation to a single site within each wing cell (Pataki, 2010).
Rab23 appears to regulate two main aspects of trichome development, hair orientation and hair number. In pupal wing cells, the absence of Rab23 leads to increased actin accumulation in the subapical region and the formation of multiple hairs. In addition, Rab23 mutations impair hexagonal packing of the wing cells, and to a lesser degree, affect cortical polarization of the PCP proteins. Although, Rab23 does not appear to exhibit a polarized distribution in wing cells, it was found that Rab23 associates with Pk, which normally accumulates in the proximal cortical domain (Pataki, 2010).
Careful comparison of the Rab23 mutant phenotype with that of the other PCP mutations reveals that the phenotypic effect of Rab23 differs from all of the known PCP genes. Most notably, Rab23 has a specific requirement in the development of one particular type of subcellular structure (i.e., the cuticular hair) in every body region examined. However, it does not appear to play any role in the planar orientation of multicellular units such as ommatidia in the eye or the sensory bristles of the adult epidermis. In contrast to this, other PCP genes typically exhibit a tissue specific, but not structure specific, requirement, or, such as mutations of the core group, affect the polarization of every tissue and structure, regardless of whether they are hairs, bristles, or unit eyes. Focusing on the wing, loss of Rab23 results in weak trichome orientation defects and a relatively strong multiple hair phenotype (mostly double hairs). This is clearly different from the core PCP phenotypes (strong hair orientation defects and few multiple hairs), or the phenotypes of the In group and mwh (strong orientation defects and multiple hairs in almost every cell). As compared to Rho1 and Drok, Rab23 displays a similar adult wing hair phenotype in mutant clones with respect to multiple hairs, while the orientation defects are less clear in Rho1 and Drok mutants than in Rab23. Moreover, a significant difference exists at the molecular level, because, unlike Rab23, Rho1 and Drok do not play a role in cortical polarization of the core PCP proteins. Given that Rab23 alleles genetically behave as strong LOF or null alleles, Rab23 identifies a unique class of PCP genes dedicated to the regulation of trichome planar polarization (Pataki, 2010).
Although some recent data suggested that the establishment of properly polarized cortical domains is not an absolute requirement for correct trichome polarity in the wing, asymmetric accumulation of the PCP proteins is thought to serve as a critical cue for cell polarization. Thus, the Rab23-induced weak alterations in wing hair polarity are best explained by the similarly modest effect on PCP protein asymmetries. Because Rab23 is able to associate with Pk, it follows that Rab23 is likely to play a role in the proximal accumulation of Pk. Given that the Rab family of proteins is known to control membrane trafficking, the results provide further support for models suggesting that polarized membrane transport is an important mechanism for the asymmetric accumulation of the PCP proteins. Although Rab23 showed a specific interaction with Pk, technical limitations might have prevented the detection of interactions with other core PCP proteins, and hence it is possible that the mechanism whereby Rab23 contributes to cortical polarization is not limited to Pk regulation. One additional candidate is the transmembrane protein Vang that partly colocalizes with Rab23 in S2 cells and has been shown to bind Pk. Thus, through binding to Pk, Rab23 might affect Vang localization or signaling capacity. Irrespective of whether Rab23 directly affects the localization of only one or more PCP proteins, in the wing Rab23 has a relatively modest effect on protein localization, and, as a consequence, on hair orientation, indicating that Rab23 has a minor or largely redundant role in this tissue. Interestingly, however, Rab23 induces much stronger trichome orientation defects on the abdominal cuticle. Although it is not proven formally, genetic analysis suggests that asymmetric PCP protein accumulation (or at least polarized activation) is likely to occur in the abdominal histoblast cells as well. Hence, with respect to protein polarization Rab23 may act in a tissue-specific manner playing a largely dispensable role in the wing, but having a critical role in the abdominal epidermis (Pataki, 2010).
Correct trichome placement at a single distally located site is clearly a crucial step in planar polarization of the wing cells. Current models suggest that prehair initiation is controlled by an inhibitory cue localized proximally in a Vang-dependent manner, and by a Fz-dependent cue that positively regulates hair formation at the distal vertex. Whereas it is not clear how the distal cues work, with regard to the proximal cues it is known that Vang and Pk colocalize with the effector proteins In, Fy and Frtz that control the localization and activity of Mwh, which is thought to regulate prehair initiation directly by interfering with actin bundling in the subapical region of cells. This study found that Rab23 severely impairs trichome placement in the wing leading to the formation of multiple hairs, which indicates a role in the repression of ectopic hair initiation. Where does Rab23 fit into the regulatory hierarchy of trichome placement? Double mutant analysis suggests that Rab23 is upstream of the In group and mwh, and acts at the same level as the core PCP genes. The synergistic genetic interaction between Rab23 and the core PCP mutations indicates that they function in parallel pathways during the restriction of prehair initiation. Remarkably, the pkpk; Rab23 double mutants exhibit an almost identical phenotype to mutations of the In group, suggesting that, unless the existence of an In independent restriction system is assumed, Pk and Rab23 together are both necessary and sufficient to fully activate the In complex. In pk single mutants the proximal accumulation of In is severely impaired, yet multiple hairs rarely develop, indicating that proper In localization plays only a minor role in the restriction mechanism. Conversely, in Rab23 single mutants In localization is weakly affected, but multiple hairs often form, suggesting that the major function of Rab23 is related to In activation. Thus, it appears that the proximally restricted activation of In on the one hand is ensured by Pk, that mainly plays a role in proper In localization, and on the other hand by Rab23, that seems to be required for In activation. At present, the molecular function of the In system is unknown, and it is therefore also unclear how Rab23 might contribute to the activation of the In complex. Nevertheless, because Rab23 has a weaker multiple hair phenotype than in, but the pkpk; Rab23 double mutant is nearly as strong as in, it is conceivable that In activation is not exclusively Rab23 dependent but, beyond a role in protein localization, Pk has a partial requirement as well (Pataki, 2010).
The regulation of cellular packing is an interesting, yet only lately appreciated aspect of wing development. It has been reported that the wing epithelium is irregularly packed throughout larval and prepupal stages, but shortly before hair formation it becomes a quasihexagonal array of cells. Hexagonal repacking depends on the activity of the core PCP proteins. However, defects in packing geometry do not appear to directly perturb hair polarity in core PCP mutant wing cells. The possible exception to this rule is pk that exhibits very strong hair orientation defects and induces the strongest packing defects within the core PCP group. Additionally, another study revealed that irregularities in cell geometry are associated with polarity defects in the case of fat mutant clones. Thus, cell geometry is not the direct determinant of cell polarity, but in some instances cell packing seems to impact on PCP signaling and hair orientation. This study has shown that in the wing Rab23 is predominantly involved in the regulation of wing hair number, and it is also required for hexagonal packing of the wing epithelium. Do these packing defects correlate with the severity of the multiple hair phenotype? The data argue against this idea for the case of Rab23, and also for the cases of other strong multiple hair mutants, such as in, frtz, and mwh. Therefore, cell shape has no direct effect on the regulation of the number of prehair initiation sites, and Rab23 appears to regulate hexagonal packing and hair number independently (Pataki, 2010).
As Rab23 and Pk are both required for cellular packing, and Rab23 associates with Pk, it is possible that they cooperate during the regulation of packing. This is in agreement with the observation that pkpk; Rab23 double mutant wings do not show stronger packing defects than a pkpk single mutant. However, other interpretations are also possible, hence further investigations will be required to understand how Rab23 and Pk regulates cellular packing and to clarify the impact of packing geometry on PCP establishment in the wing (Pataki, 2010).
Unlike the vertebrate orthologs, Drosophila Rab23 is not an essential gene and does not appear to regulate Hedgehog signaling. Given that Rab GTPases are thought to regulate vesicular transport and that mouse Rab23 localizes to endosomes, it was expected that Rab23 regulates the trafficking of vesicle-associated Hedgehog signaling components. However, in the mammalian systems no clear link between endocytosis, Rab23, and the subcellular localization of Hedgehog signaling elements has been identified. The finding that Rab23 associates with Pk suggests that Rab23 might be directly involved in the regulation of Pk trafficking, and therefore Pk could be the first known direct target of Rab23. Interestingly, there is a significant overlap reported in the embryonic expression domains of the vertebrate Pk and Rab23 genes in the region of the dorsal neural ectoderm, the somites, and the limb buds. Moreover, it is also known that blocking of Rab23 or Pk function in vertebrate embryos can both lead to a spina bifida phenotype. These observations raise the possibility that, unlike the Rab23 involvement in Hedgehog signaling, the Rab23-Pk regulatory connection is evolutionarily conserved (Pataki, 2010).
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